The sensitive measurement of nucleic acids (NAs) is important in a number of fields such as clinical diagnostics, environmental monitoring, detection of biological threats, and food safety. The detection and quantification of NAs at low concentrations from viruses and bacteria is especially important for the early diagnosis of infectious diseases and testing of blood supplies.
The gold standard method for detecting DNA is the polymerase chain reaction (PCR). PCR is now capable of routinely detecting <100 copies of NA per sample (volume ˜5 μL), and real time PCR assays for HIV, for example, are capable of detecting down to 20 copies of viral RNA per mL. PCR is based on the exponential amplification of target molecules by repeated cycles of molecular replication. PCR has proven to be robust as well as sensitive, and has found widespread use in clinical diagnostics and research. PCR does, however, have drawbacks—primarily from the reliance on polymerases—that limit its usefulness in some applications. First, polymerases are susceptible to inhibition from sample components that can result in false negative results in clinical, environmental, food, and forensic samples. As a result, PCR requires careful sample preparation to purify DNA in order for the amplification reaction to be performed. DNA purification makes automation of PCR more complex, with each type of sample often requiring a different preparation method. Second, PCR can be prone to false positive results from erroneous amplification of non-target sequences. Third, PCR is relatively expensive compared to other sensitive analytical techniques, such as immunoassays. Methods that do not rely on the replication of DNA, i.e., that directly detect the target molecules without polymerases, are promising alternatives to PCR.
Accordingly, improved methods are needed.